Representation of unique sequences in libraries of randomized nucleic acids

Abstract

From a library of nucleic acid molecules, which are randomized in parts of their sequence, unique sequence variants can be selected for specific properties. The planning of such an in vitro selection experiment requires some consideration regarding how much DNA template or RNA transcript should be used initially. The amount applied depends on the number of randomized nucleotides and on the expectations of how often each conceivable and unique sequence combination should be represented in the experimental pool. We display graphs describing the probability for the representation of unique nucleic acid molecules in a randomized pool as a function of the mean representation k, defined by the ratio of sampled nucleic acid molecules to conceivable sequence combinations and we summarize the amounts required to represent unique sequences with 99% likelihood. The probability of representation, P = 1 - e(-k) can be applied also to 'sub-saturated' pools (k < 1) of nucleic acids with long randomized domains, where it is impossible to provide sufficient material for full sequence representation.